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Essential and unique roles of PIP5K-gamma and -alpha in Fcgamma receptor-mediated phagocytosis.

Mao YS, Yamaga M, Zhu X, Wei Y, Sun HQ, Wang J, Yun M, Wang Y, Di Paolo G, Bennett M, Mellman I, Abrams CS, De Camilli P, Lu CY, Yin HL - J. Cell Biol. (2009)

Bottom Line: The actin cytoskeleton is dynamically remodeled during Fcgamma receptor (FcgammaR)-mediated phagocytosis in a phosphatidylinositol (4,5)-bisphosphate (PIP(2))-dependent manner.Delivery of exogenous PIP(2) rescued these defects.In addition, PIP5K-gamma but not PIP5K-alpha is transiently activated by spleen tyrosine kinase-mediated phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

ABSTRACT
The actin cytoskeleton is dynamically remodeled during Fcgamma receptor (FcgammaR)-mediated phagocytosis in a phosphatidylinositol (4,5)-bisphosphate (PIP(2))-dependent manner. We investigated the role of type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) gamma and alpha isoforms, which synthesize PIP(2), during phagocytosis. PIP5K-gamma-/- bone marrow-derived macrophages (BMM) have a highly polymerized actin cytoskeleton and are defective in attachment to IgG-opsonized particles and FcgammaR clustering. Delivery of exogenous PIP(2) rescued these defects. PIP5K-gamma knockout BMM also have more RhoA and less Rac1 activation, and pharmacological manipulations establish that they contribute to the abnormal phenotype. Likewise, depletion of PIP5K-gamma by RNA interference inhibits particle attachment. In contrast, PIP5K-alpha knockout or silencing has no effect on attachment but inhibits ingestion by decreasing Wiskott-Aldrich syndrome protein activation, and hence actin polymerization, in the nascent phagocytic cup. In addition, PIP5K-gamma but not PIP5K-alpha is transiently activated by spleen tyrosine kinase-mediated phosphorylation. We propose that PIP5K-gamma acts upstream of Rac/Rho and that the differential regulation of PIP5K-gamma and -alpha allows them to work in tandem to modulate the actin cytoskeleton during the attachment and ingestion phases of phagocytosis.

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PIP5K-γ−/− BMM are defective in attachment to IgG-opsonized particles and response to IC. (A) Particle attachment and ingestion. (left) Fluorescence/DIC images. DIC images were overlaid on the fluorescence images in the “ingestion” panels. Green, external beads; red, phalloidin. (top right) Phagocytic indices, defined as the mean number of beads per cell (n > 150 cells). (bottom right) Particle attachment histogram. (B) IC-induced FcγR microclustering. BMM incubated with IC at 4°C were stained with anti-IgG to detect FcγR clusters on the cell surface. (left) Fluorescence images. (right) Cluster size quantitation by ImageJ (n = ∼10,000 clusters). Size was expressed in arbitrary units (A.U.) relative to WT BMM at 5 min. Bars, 10 µm. (C) IC-induced actin depolymerization. Polymerized actin was quantitated using a fluorometric phalloidin-binding assay. The ratio of FITC-phalloidin to DAPI intensity was expressed as the percentage of WT BMM without IC (n = 6). (D) IC-induced ERK phosphorylation. BMM were exposed to IC at 4°C for 20 min, washed, and incubated at 37°C for the times indicated. ERK and pERK were detected by Western blotting. (E) IC-induced change in PIP2 homeostasis. BMM were challenged with or without IC for 2 min at 37°C. The PIP2 level was quantitated by HPLC and expressed as the percentage of WT BMM without IC treatment (n = 3). Error bars indicate SEM.
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fig2: PIP5K-γ−/− BMM are defective in attachment to IgG-opsonized particles and response to IC. (A) Particle attachment and ingestion. (left) Fluorescence/DIC images. DIC images were overlaid on the fluorescence images in the “ingestion” panels. Green, external beads; red, phalloidin. (top right) Phagocytic indices, defined as the mean number of beads per cell (n > 150 cells). (bottom right) Particle attachment histogram. (B) IC-induced FcγR microclustering. BMM incubated with IC at 4°C were stained with anti-IgG to detect FcγR clusters on the cell surface. (left) Fluorescence images. (right) Cluster size quantitation by ImageJ (n = ∼10,000 clusters). Size was expressed in arbitrary units (A.U.) relative to WT BMM at 5 min. Bars, 10 µm. (C) IC-induced actin depolymerization. Polymerized actin was quantitated using a fluorometric phalloidin-binding assay. The ratio of FITC-phalloidin to DAPI intensity was expressed as the percentage of WT BMM without IC (n = 6). (D) IC-induced ERK phosphorylation. BMM were exposed to IC at 4°C for 20 min, washed, and incubated at 37°C for the times indicated. ERK and pERK were detected by Western blotting. (E) IC-induced change in PIP2 homeostasis. BMM were challenged with or without IC for 2 min at 37°C. The PIP2 level was quantitated by HPLC and expressed as the percentage of WT BMM without IC treatment (n = 3). Error bars indicate SEM.

Mentions: WT BMM attached to coverslips were predominantly polygonal, whereas PIP5K-γ−/− BMM were often elongated and stellate (Fig. 2 A). PIP5K-γ−/− BMM also had brighter phalloidin staining, especially in the cell cortex. The bulk fluorometric phalloidin assay showed that there was a 50% increase in polymerized actin (Fig. 2 C). In addition, they phagocytized fewer IgG-opsonized particles (Fig. 2 A). Using incubation at 4°C versus 37°C to separate particle attachment (which can occur at 4°C) from engulfment (which does not occur at 4°C), we found that there was a marked decrease in particle binding (Fig. 2 A). The binding index (mean number of attached particles per cell) decreased by 70%, and the binding histogram shifted to the left (Fig. 2 A). The decrease in particle attachment paralleled that of ingestion, which suggests that the primary defect resides in the initial attachment phase. Similar inhibition was observed using IgG-opsonized sheep red blood cells (unpublished data).


Essential and unique roles of PIP5K-gamma and -alpha in Fcgamma receptor-mediated phagocytosis.

Mao YS, Yamaga M, Zhu X, Wei Y, Sun HQ, Wang J, Yun M, Wang Y, Di Paolo G, Bennett M, Mellman I, Abrams CS, De Camilli P, Lu CY, Yin HL - J. Cell Biol. (2009)

PIP5K-γ−/− BMM are defective in attachment to IgG-opsonized particles and response to IC. (A) Particle attachment and ingestion. (left) Fluorescence/DIC images. DIC images were overlaid on the fluorescence images in the “ingestion” panels. Green, external beads; red, phalloidin. (top right) Phagocytic indices, defined as the mean number of beads per cell (n > 150 cells). (bottom right) Particle attachment histogram. (B) IC-induced FcγR microclustering. BMM incubated with IC at 4°C were stained with anti-IgG to detect FcγR clusters on the cell surface. (left) Fluorescence images. (right) Cluster size quantitation by ImageJ (n = ∼10,000 clusters). Size was expressed in arbitrary units (A.U.) relative to WT BMM at 5 min. Bars, 10 µm. (C) IC-induced actin depolymerization. Polymerized actin was quantitated using a fluorometric phalloidin-binding assay. The ratio of FITC-phalloidin to DAPI intensity was expressed as the percentage of WT BMM without IC (n = 6). (D) IC-induced ERK phosphorylation. BMM were exposed to IC at 4°C for 20 min, washed, and incubated at 37°C for the times indicated. ERK and pERK were detected by Western blotting. (E) IC-induced change in PIP2 homeostasis. BMM were challenged with or without IC for 2 min at 37°C. The PIP2 level was quantitated by HPLC and expressed as the percentage of WT BMM without IC treatment (n = 3). Error bars indicate SEM.
© Copyright Policy - openaccess
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC2654300&req=5

fig2: PIP5K-γ−/− BMM are defective in attachment to IgG-opsonized particles and response to IC. (A) Particle attachment and ingestion. (left) Fluorescence/DIC images. DIC images were overlaid on the fluorescence images in the “ingestion” panels. Green, external beads; red, phalloidin. (top right) Phagocytic indices, defined as the mean number of beads per cell (n > 150 cells). (bottom right) Particle attachment histogram. (B) IC-induced FcγR microclustering. BMM incubated with IC at 4°C were stained with anti-IgG to detect FcγR clusters on the cell surface. (left) Fluorescence images. (right) Cluster size quantitation by ImageJ (n = ∼10,000 clusters). Size was expressed in arbitrary units (A.U.) relative to WT BMM at 5 min. Bars, 10 µm. (C) IC-induced actin depolymerization. Polymerized actin was quantitated using a fluorometric phalloidin-binding assay. The ratio of FITC-phalloidin to DAPI intensity was expressed as the percentage of WT BMM without IC (n = 6). (D) IC-induced ERK phosphorylation. BMM were exposed to IC at 4°C for 20 min, washed, and incubated at 37°C for the times indicated. ERK and pERK were detected by Western blotting. (E) IC-induced change in PIP2 homeostasis. BMM were challenged with or without IC for 2 min at 37°C. The PIP2 level was quantitated by HPLC and expressed as the percentage of WT BMM without IC treatment (n = 3). Error bars indicate SEM.
Mentions: WT BMM attached to coverslips were predominantly polygonal, whereas PIP5K-γ−/− BMM were often elongated and stellate (Fig. 2 A). PIP5K-γ−/− BMM also had brighter phalloidin staining, especially in the cell cortex. The bulk fluorometric phalloidin assay showed that there was a 50% increase in polymerized actin (Fig. 2 C). In addition, they phagocytized fewer IgG-opsonized particles (Fig. 2 A). Using incubation at 4°C versus 37°C to separate particle attachment (which can occur at 4°C) from engulfment (which does not occur at 4°C), we found that there was a marked decrease in particle binding (Fig. 2 A). The binding index (mean number of attached particles per cell) decreased by 70%, and the binding histogram shifted to the left (Fig. 2 A). The decrease in particle attachment paralleled that of ingestion, which suggests that the primary defect resides in the initial attachment phase. Similar inhibition was observed using IgG-opsonized sheep red blood cells (unpublished data).

Bottom Line: The actin cytoskeleton is dynamically remodeled during Fcgamma receptor (FcgammaR)-mediated phagocytosis in a phosphatidylinositol (4,5)-bisphosphate (PIP(2))-dependent manner.Delivery of exogenous PIP(2) rescued these defects.In addition, PIP5K-gamma but not PIP5K-alpha is transiently activated by spleen tyrosine kinase-mediated phosphorylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.

ABSTRACT
The actin cytoskeleton is dynamically remodeled during Fcgamma receptor (FcgammaR)-mediated phagocytosis in a phosphatidylinositol (4,5)-bisphosphate (PIP(2))-dependent manner. We investigated the role of type I phosphatidylinositol 4-phosphate 5-kinase (PIP5K) gamma and alpha isoforms, which synthesize PIP(2), during phagocytosis. PIP5K-gamma-/- bone marrow-derived macrophages (BMM) have a highly polymerized actin cytoskeleton and are defective in attachment to IgG-opsonized particles and FcgammaR clustering. Delivery of exogenous PIP(2) rescued these defects. PIP5K-gamma knockout BMM also have more RhoA and less Rac1 activation, and pharmacological manipulations establish that they contribute to the abnormal phenotype. Likewise, depletion of PIP5K-gamma by RNA interference inhibits particle attachment. In contrast, PIP5K-alpha knockout or silencing has no effect on attachment but inhibits ingestion by decreasing Wiskott-Aldrich syndrome protein activation, and hence actin polymerization, in the nascent phagocytic cup. In addition, PIP5K-gamma but not PIP5K-alpha is transiently activated by spleen tyrosine kinase-mediated phosphorylation. We propose that PIP5K-gamma acts upstream of Rac/Rho and that the differential regulation of PIP5K-gamma and -alpha allows them to work in tandem to modulate the actin cytoskeleton during the attachment and ingestion phases of phagocytosis.

Show MeSH
Related in: MedlinePlus